The dramatic and ever-increasing emergence of medically relevant strains of bacteria resistant to traditional antibiotics is now a major human health issue. Antibiotic resistance has arisen due to the extensive clinical use of classical antibiotics. We have now seen the development of so-called ?Superbugs? that are resistant to most or all of the available antibiotics. The Infectious Diseases Society of America has reported that two-thirds of all health-care associated infections are caused by six multi-drug resistant organisms referred to as ?ESKAPE? pathogens. The proposed project focuses on development of novel peptide antibiotics for two Gram-negative ESKAPE organisms (Acinetobacter baumannii and Pseudomonas aeruginosa). Compared to existing antibiotics, antimicrobial peptides (AMPs) are a radically different structural class of antibiotics. They kill target cells rapidly, have broad-spectrum activity and are active against serious antibiotic-resistant pathogens isolated in clinics. The major barrier to the use of native AMPs as antibiotics has been their toxicity or ability to lyse eukaryotic cells. Dr. Hodges laboratory made a major breakthrough in AMP research with the discovery that incorporation of ?specificity determinants? into existing AMPs, whether native or de novo designed, can enhance specific antimicrobial activity and eliminate toxicity to human red blood cells and or other eukaryotic cells. For example, the therapeutic index of our compound D16 was improved 1305-fold against A. baumannii and 895-fold against P. aeruginosa compared to the starting broad-spectrum native AMP compound. In addition, we were able to convert a native AMP, D-dermaseptin S4, with broad spectrum activity and high toxicity to human red blood cells to D-dermaseptin S4 (L7K, A14K) with Gram-negative selectivity and a 730-fold and 980-fold improvement in therapeutic indices against these same gram-negative pathogens, respectively. Our all D-conformation peptides are completely resistant to proteolysis and show excellent antimicrobial activity in the presence of serum proteins (two critical properties for moving our pre-clinical candidates forward in the development process). As our compounds are membrane active peptides whose sole target is the cytoplasmic membrane of the pathogen, through non-specific interactions, development of bacterial resistance is unlikely, since this would require substantial changes in the lipid composition of microbial cell membrane. Our AMPs are all active against colistin and polymyxin B (antibiotics of last resort) resistant strains of A. baumannii. We have formed the AMP Discovery Division of Aurora Oncology Inc. to carry out the development of three lead compounds, D16, D26 and D-dermaseptin S4 (L7K, A14K), with 4 other compounds in reserve. The company has licensed the technology from the University of Colorado (extensive patent portfolio with 3 issued patents, one pending and one to be filed). We have demonstrated efficacy in preliminary rat models. The proposed milestone-driven Phase I project is focused on confirming efficacy in a larger rat study and determining safety and pharmacokinetics of the compounds as rapidly as possible.